Name: a murine model of irreversible and reversible unilateral ureteric obstruction
Uploaded: 2014-12-20T15:00:00.000+00:00
Duration: 14 min 5 s
Description: Watch this Scientific Journal Video about A Murine Model of Irreversible and Reversible Unilateral Ureteric Obstruction at JoVE.com

The present study was supported by grants from Kidney Research UK (ST4/2011), the Cunningham Trust (CT11/14), the Mrs AE Hogg Charitable Trust for Kidney Research and the Renal Endowment Fund of the Royal Infirmary of Edinburgh.

Guía General: Los experimentos con animales se realizaron de acuerdo con los lineamientos y regulaciones impuestas por los Animales (Procedimientos Científicos) Ley de 1986. Este protocolo y el protocolo de vídeo que acompaña son tanto para una OUU estándar y un R-OUU, que se puede realizar en muchos cepas de ratón. En el video que acompaña, ambos procedimientos se llevan a cabo en ratones machos C57BL / 6 ratones de 8 semanas. Los datos presentados en la sección de resultados representativos se obtuvieron de machos FVB / n ratones. NOTA: Este protocolo y acompañando detalles del video de cómo realizar una OUU estándar y R-OUU utilizando el uréter izquierdo, sin embargo las mismas técnicas pueden ser fácilmente aplicadas al uréter derecho. 1. Preparación de los animales y laparotomía <ol><li> Realice todos los procedimientos con instrumentos estériles (en autoclave) y consumibles. </li><li> Inyectar clorhidrato de ketamina (70 mg / kg) y clorhidrato de medetomidina (1 mg / kg) por vía intraperitoneal para anestesiarel ratón. Nota: La duración del plano anestésico resultante es de 4 horas y no se requiere anestesia suplementaria. </li><li> Confirme la profundidad de la anestesia por la pérdida de los reflejos por ejemplo, pizca dedo del pie. </li><li> Eliminar todo el pelo que rodea el área de la incisión y preparar la piel para la cirugía abdominal a través de la aplicación de una solución de clorhexidina diluido. </li><li> Coloque el ratón sobre una almohadilla quirúrgica climatizada en posición supina y fijar las extremidades a la almohadilla con cinta adhesiva de baja adherencia. </li><li> Durante el procedimiento, supervisar el ratón para detectar signos de quemaduras térmicas como resultado de la almohadilla quirúrgica calentada. Si es posible utilizar una fuente de calor no eléctrico. </li><li> Administrar analgésico mediante una inyección subcutánea de hidrocloruro de buprenorfina (0.06mg / kg) y aplicar lubricante ocular para evitar el secado de la córnea. </li><li> Hacer una laparotomía media y una incisión de la línea alba avascular utilizando tejido que separa las tijeras para obtener acceso a la cavidad peritoneal. </li><li> Coloque el ratón e inserte un colibriretractor en la incisión. </li></ol> 2. Unilateral ureteral Obstrucción <ol><li> El uso de bastoncillos de algodón esterilizados exponer el uréter izquierdo desplazando los intestinos hacia el lado derecho de la cavidad abdominal y cubre con paños humedecidos. </li><li> Con unas pinzas en ángulo aislar y levantan el uréter izquierdo. </li><li> Para crear una obstrucción de la uretra, ligar el uréter izquierdo dos veces con 6 / O trenzado negro sutura de seda en cualquier lugar entre la vejiga y pelvis renal. Para los experimentos a largo plazo, el uso de sutura absorbible para todas las cirugías abdominales. Alternativamente, un clip de ligadura se puede aplicar con el uréter. </li><li> Para aislar la vejiga al uréter, divida el uréter entre los dos puntos de sutura. </li><li> Vuelva a colocar cuidadosamente los intestinos a la cavidad peritoneal. </li><li> Siga los pasos que se indican en la Sección 4 - Recuperación Post-Operativo y Cuidado para cerrar la incisión y revertir la anestesia. </li></ol> 3. Preparación para Reversible unilateral ureteral Obstrucción <ol><li> Preparar un ratón para la cirugía y aislar el uréter izquierdo tal como se detalla en los pasos 1.1 a 2.2 anteriormente. </li><li> Para crear una obstrucción de la uretra que puede ser revertida, ligar el uréter izquierdo dos veces con 6 / O trenzado negro sutura de seda cerca de la vejiga. Deja un extremo de la sutura superior siempre y cuando este se utiliza para anclar el tubo de plástico de pared suave en su lugar alrededor del uréter. </li><li> Hacer una hendidura longitudinal en una longitud de 5 mm de tubo de plástico de pared blanda, que puede ser extendió abierta para permitir que se aplique con el uréter. Cualquier tubo de plástico blando de silicona amurallada, con un diámetro interno de 1 mm y un diámetro exterior de 2 mm, se puede utilizar. </li><li> Coloque el tubo de plástico de paredes suaves suavemente alrededor del uréter. Asegúrese de que el largo de la sutura emerge desde el centro de la ranura en el tubo una vez que se cierra alrededor del uréter. </li><li> Coloque una longitud de 6 / O trenzado negro sutura de seda alrededor de la tubería de pared suave que encierra el uréter y ataruna vez. Ahora coloque el extremo largo de la sutura, emergiendo desde el centro de la ranura en el tubo, longitudinalmente a través de la tubería de manera que se sitúe en la parte superior de la sutura previamente atado. </li><li> Ate la sutura situado alrededor del tubo dos veces para anclar la tubería y el extremo largo de sutura en su lugar alrededor de la uretra. Nota: Para evitar la formación de adherencias en el uréter y el tubo de plástico de pared suave, una solución de reducción de la adhesión se puede aplicar a la zona alrededor de la tubería. </li><li> Vuelva a colocar cuidadosamente los intestinos a la cavidad peritoneal. </li></ol> Recuperación y Cuidados 4. Post-Operativo <ol><li> Cierre del peritoneo con puntada manta usando 5 / O trenzado negro sutura de seda y aproximar la piel con clips metálicos de la piel. </li><li> Para minimizar el riesgo de infección post-operatoria, aplicar un antiséptico tal como una solución de yodo / alcohol a la piel abdominal. </li><li> Parcialmente anestesia atrás con clorhidrato de atipamezol (2 mg / kg) por vía subcutánea. </li&gt; <li> Administrar fluidos por una inyección subcutánea de 1 ml de solución salina calentada. </li><li> Monitorear el ratón hasta que se haya recuperado la conciencia. </li><li> Deje que el ratón se recupere en una caja climatizada mantenido a 29 ° C durante 24 horas. Comida humedecida también se puede proporcionar para estimular la ingesta de líquidos y la nutrición. </li><li> Deje el ratón para recuperar para inducir un nivel deseado de obstrucción, típicamente de 7 días. </li><li> Para los experimentos de recuperación a largo plazo, proporcionar analgésicos en curso. Si el ratón se ha de recuperar por más de 7 días, retire los clips de la piel 7 días después de la cirugía. </li><li> Una vez que el nivel deseado de obstrucción ha sido inducida, ya sea invertir la OUU, como se describe a continuación, o la eutanasia el ratón por dislocación cervical y recoger los riñones para el análisis de la histopatología. </li></ol> 5. reversible unilateral ureteral Obstrucción <ol><li> Para realizar una R-OUU preparar un ratón, que ha sido objeto de preparación para R-OUU, para la cirugía como se describe en los pasos 10,1-1,7. </li><li> Si está presente, quitar los clips de la piel y dividir o eliminar las suturas en el peritoneo para obtener acceso a la cavidad abdominal. Nota: Si el uréter ha sido obstruido por un largo plazo clips piel experimento debería haber sido eliminado 7 días después de la aplicación. </li><li> Preparar el ratón y aislar el uréter izquierdo tal como se describe en los pasos 1.8 a 2.2. </li><li> Con unas pinzas en ángulo, liberar el tubo de plástico de paredes blandas de cualquier tejido granulomatoso que se hayan formado. </li><li> Cortar la sutura sujeta el tubo de plástico alrededor del uréter obstruido con un bisturí y retire el tubo. </li><li> Confirme éxito OUU mediante la evaluación de la presencia de hidronefrosis en el riñón izquierdo, el riñón también debería aparecer pálido. </li><li> Divida el uréter entre las suturas. </li><li> Coloque la longitud restante del uréter, que se adjunta en el riñón, en un pequeño trozo de gasa estéril. Esto será utilizado para recoger el sedimento urinario y las células muertas que drenará desde el uréter unand pelvis renal una vez que la sutura se retira. </li><li> Divida el uréter por encima, pero cerca, la sutura y permitir que el riñón para drenar en la gasa. Deja la sutura inferior, más cercana a la vejiga, intacto. Esto es para asegurar que no tendrá fugas de orina desde la vejiga y en la cavidad peritoneal. </li><li> Una vez que el uréter y pelvis renal han sido drenados, aplicar un largo 6 / O negro sutura de seda trenzada hasta el final de la longitud restante del uréter. Esto se utiliza para ayudar el uréter a la vejiga anastomosis realizada más tarde. </li><li> Siga los pasos de abajo para anastomose la longitud restante de uréter en la vejiga: <ol><li> Girar el uréter manera que se sitúe anterior a su posición original y se encuentra sobre el riñón. </li><li> Coloque un solo 9 / O monofilamento de poliamida viradas de sutura 2 mm desde el extremo del uréter tal que emerge en la dirección de la vejiga. Tener cuidado para asegurar que la sutura permanece en la capa muscular y no entra en el lumen del uréter. El wil sutura viradasl se utiliza para anclar el uréter en la vejiga. </li><li> Para crear un canal a través de la vejiga, pasar una aguja 21G en diagonal a través de la vejiga, tales que sale hacia el frontal (ventral) de la pared de la vejiga. </li><li> Descanse una aguja de ojos en el bisel de la aguja 21G. Utilice la aguja 21G para guiar la aguja de ojos a través de la vejiga. La aguja de ojos se utilizará para tomar el uréter a través de la vejiga. </li><li> Pasar el viradas de sutura 9 / O, aplicado con el uréter en el paso 5.11.2, a través de la primera incisión en la vejiga y hacia fuera a través de la pared de la vejiga adyacente al punto de entrada. Una vez que el uréter se pasa a través de la vejiga, esto será atado para anclar el uréter a la vejiga. </li><li> Coloque el largo 6 / O sutura de seda trenzada negro aplicada al extremo del uréter en el paso 5.10 través de la aguja de ojos. </li><li> Retirar cuidadosamente la aguja de ojos fuera de la vejiga garantizando al mismo tiempo que el uréter también se tira a través de la vejiga. </li><li> Una vez que el uréter emerge de The vejiga retire la aguja de ojos y aplicar una abrazadera a la larga de sutura en el extremo del uréter para evitar la retracción de nuevo en la vejiga. </li><li> Para anclar el uréter dentro de lugar en la vejiga, atar el 9 / O de poliamida de sutura monofilamento de pegajosidad, aplicada en el paso 5.11.5. </li><li> Aplicar individuales 9 / O poliamida suturas lazo monofilamento adicionales como se describe en los pasos 5.11.2 y 5.11.5 en dos localidades de todo el punto de entrada para anclar firmemente el uréter en la vejiga. </li><li> Con el dorso de tijeras abiertas, empuje la pared de la vejiga hacia atrás ligeramente para exponer más del uréter emergente. </li><li> Divida el uréter por encima de la sutura larga situada en el extremo del uréter. El uréter debe retraerse en el cuerpo de la vejiga. Es habitual observar orina que fluye fuera de la abertura en la vejiga, lo que confirma el lumen abiertos del uréter. </li><li> Cierre el orificio de salida en la vejiga con un monofilamento de poliamida de sutura 9 / O. </li></ol></li><li> Vuelva a colocar cuidadosamente los intestinos enla cavidad peritoneal. </li><li> Proporcionar cuidados postoperatorios, como se detalla en los pasos 4.1 a 4.8. </li><li> Deje que el ratón para recuperar hasta que se descomprime el riñón, por lo general 7 días. </li><li> En el punto final experimental, la eutanasia el ratón por dislocación cervical y recoger los riñones para el análisis histopatológico. </li></ol>

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Obstruction of the kidney may affect native or transplanted kidneys and results in kidney injury and scarring. The R-UUO model is of relevance to human disease as patients often present with obstruction of the renal tract secondary to prostatic hypertrophy, posterior urethral valves etc. that requires decompression with patients commonly left with significant residual kidney impairment that has no current treatment options and may eventually lead to end stage kidney disease.8,9 The models detailed here enable ...

The overall aim of the experimental model described here is to induce obstructive nephropathy by unilateral ureteric obstruction (UUO), which can either be irreversible (UUO) or reversible (R-UUO). A simple irreversible model of UUO is presented in which the left ureter is permanently obstructed by ligation with a suture or by the application of a ligating clip. This results in marked dilatation of the ureter together with reduced renal blood flow and glomerular filtration. Renal histology demonstrates tubular dilatation and increasingly severe interstitial renal inflammation and fibrosis. Irreversible UUO is a useful model and has been adopted by many researchers in the study of both renal inflammation and fibrosis.1-4 Although the irreversible model of UUO requires some surgical expertise it is relatively straightforward and is often used to seek insights into the pathogenesis of interstitial renal injury and the ensuing fibrosis. Also presented is a less frequently used R-UUO model using a modification of the method originally described by Tapmeier et al.5 The R-UUO model has much future potential for the study of inflammatory and immune processes, cellular and tissue regeneration as well as the subsequent tissue remodeling following the removal of an injurious stimulus. 
The more challenging R-UUO model has been used by a limited number of investigators with some groups employing a significantly different surgical technique to that described here6,7 though with interesting results. In the R-UUO model presented, the ureter is ligated to induce complete ureteric obstruction for a period of time sufficient to induce the level of injury and fibrosis desired: 7 days of UUO was chosen in the method described here. The ureteric obstruction is reversed and the kidney is allowed to decompress for a period of time determined by the investigator before the mice are culled and the kidney removed for analysis: 7 days of decompression was chosen in the method described here though a longer period would be chosen if the resolution of inflammation and fibrosis was being studied. Although the method described here requires significant surgical expertise, it offers several advantages over other R-UUO models. The application of soft walled plastic tubing to the obstructed ureter prevents excessive ureteric dilation and this facilitates the subsequent manipulation and anastomosis of the ureter. Furthermore, in the described R-UUO model the ureter is divided thereby allowing the removal of any residual urinary sediment and debris. This confirms that the remaining ureter lumen is de-obstructed and patent prior to anastomosis to the bladder. 
Experiments incorporating both the irreversible and reversible UUO models can provide researchers with a powerful insight into the molecular and cellular mechanisms of both injury and subsequent resolution and regeneration. Thus, the model of R-UUO described here would be highly relevant to researchers interested in post-inflammatory tissue remodeling and how this can be modulated.

<table><tbody><tr><td>Blunt Dissecting Scissors</td><td>Fine Science Tools</td><td>14072-10</td><td></td></tr><tr><td>Spring Scissors &amp;ndash; straight</td><td>Fine Science Tools</td><td>15000-10</td><td></td></tr><tr><td>Toothed forceps</td><td>Fine Science Tools</td><td>11021-12</td><td></td></tr><tr><td>Angled forceps x 2</td><td>Fine Science Tools</td><td>00649-11</td><td></td></tr><tr><td>Straight forceps</td><td>Fine Science Tools</td><td>00632-11</td><td></td></tr><tr><td>Colibri 3 cm wire retractor</td><td>Fine Science Tools</td><td>17000-03</td><td></td></tr><tr><td>Castroviejo needle holder with lock</td><td>Fine Science Tools</td><td>12565-14</td><td></td></tr><tr><td>Wound clip applicator</td><td>Fine Science Tools</td><td>12031-07</td><td></td></tr><tr><td>7 mm wound clips</td><td>Fine Science Tools</td><td>12032-07</td><td></td></tr><tr><td>Castroviejo micro needle holder with lock</td><td>Fine Science Tools</td><td>12060-01</td><td></td></tr><tr><td>OPMI pico microscope</td><td>Carl Zeiss</td><td>S100</td><td></td></tr><tr><td>Heat electronic pad</td><td>Cozee Comfort</td><td>n/a</td><td></td></tr><tr><td>6/O silk braided suture</td><td>Harvard Apparatus</td><td>72-3287</td><td></td></tr><tr><td>9/O Dafilon (polyamide) suture</td><td>B-Braun</td><td>G1111434</td><td></td></tr><tr><td>5/O braided silk suture</td><td>Harvard Apparatus</td><td>51-7680</td><td></td></tr><tr><td>Regular bevel needle, 1 inch, 21G</td><td>Becton, Dickinson and Company</td><td>305175</td><td></td></tr><tr><td>1 ml syringe slip tip</td><td>Becton, Dickinson and Company</td><td>300184</td><td></td></tr><tr><td>Wypall paper swabs</td><td>Kimberley-Clark</td><td>L40</td><td>Sterilised (Autoclave)</td></tr><tr><td>Cotton wool buds</td><td>Johnson and Johnson</td><td>n/a</td><td>Sterilised (Autoclave)</td></tr><tr><td>Plain drapes</td><td>Guardian</td><td>CB03</td><td>Sterilised (Autoclave)</td></tr><tr><td>Soft wall silicone rubber tubing</td><td></td><td></td><td>Silicone tubing - internal diameter 1.0 mm; external diameter 2.0 mm; wall thickness 0.6 mm</td></tr><tr><td>(Lacri-Lube) White soft paraffin 57.3%, mineral oil 42.5% and lanolin alcohols 0.2%</td><td>Allergan Ltd</td><td>21956GB10X</td><td></td></tr><tr><td>(Videne) Povidone-iodine 10%</td><td>Ecolab Ltd</td><td>PL 04509/0041</td><td></td></tr><tr><td>(Vetalar V) Ketamine hydrochloride</td><td>Pfizer Animal Health</td><td>Vm 42058/4165</td><td>100mg/ml solution</td></tr><tr><td>(Domitor) Medetomidine hydrochloride</td><td>Orion Pharma</td><td>Vm 06043/4003</td><td>1mg/ml</td></tr><tr><td>(Vetergesic) Bupernorphine hydrochloride</td><td>Alsto Animal Health</td><td>Vm 00063/4002</td><td>0.3mg/ml</td></tr><tr><td>(Antisedan) Atipamezole hydrochoride</td><td>Orion Pharma</td><td>Vm 06043/4004</td><td>5mg/ml</td></tr><tr><td>(Adept) 4% Icodextrin</td><td>Baxter</td><td></td><td>Adhesion reduction solution</td></tr><tr><td>NaCl 0.9%</td><td>Baxter</td><td>FKE1323</td><td></td></tr></tbody></table>

a murine model of irreversible and reversible unilateral ureteric obstruction

Obstruction of the kidney may affect native or transplanted kidneys and results in kidney injury and scarring. Presented here is a model of obstructive nephropathy induced by unilateral ureteric obstruction (UUO), which can either be irreversible (UUO) or reversible (R-UUO). In the irreversible UUO model, the ureter may be obstructed for variable periods of time in order to induce increasingly severe renal inflammation and interstitial fibrotic scarring. In the reversible R-UUO model the ureter is obstructed to induce hydronephrosis, tubular dilation and inflammation. After a suitable period of time the ureteric obstruction is then surgically reversed by anastomosis of the severed previously obstructed ureter to the bladder in order to allow complete decompression of the kidney and restoration of urinary flow to the bladder. The irreversible UUO model has been used to investigate various aspects of renal inflammation and scarring including the pathogenesis of disease and the testing of potential anti-inflammatory or anti-fibrotic therapies. The more challenging model of R-UUO has been used by some investigators and does offer significant research potential as it allows the study of inflammatory and immune processes and tissue remodeling in an injured and scarred kidney following the removal of the injurious stimulus. As a result, the R-UUO model offers investigators the opportunity to explore the resolution of kidney inflammation together with key aspects of tissue repair. These experimental models are of relevance to human disease as patients often present with obstruction of the renal tract that requires decompression and are commonly left with significant residual kidney impairment that has no current treatment options and may lead to eventual end stage kidney failure.

La aparición de los cambios renales notablemente tras obstrucción ureteral y se convierte en más pálido y tenso a la palpación con el tiempo (Figura 1). Hay una creciente dilatación del uréter proximal a la obstrucción y la pelvis renal. El riñón se vuelve cada vez atrófica como la duración de los aumentos de nefropatía obstructiva con obstrucción prolongada que resulta en una marcada adelgazamiento de la corteza renal y médula. A raíz de la inversión de la obstrucción de la uretra, el...

Watch this Scientific Journal Video about A Murine Model of Irreversible and Reversible Unilateral Ureteric Obstruction at JoVE.com

A Murine Model of Irreversible and Reversible Unilateral Ureteric Obstruction

The murine model of irreversible unilateral ureteric obstruction (UUO) is presented together with the model of reversible UUO in which the ureteric obstruction is relieved by anastomosis of the severed ureter into the bladder. These models enable the study of renal inflammation and scarring as well as tissue remodeling.

Un modelo murino de irreversible y reversible obstrucción ureteral unilateral

Obstruction of the kidney may affect native or transplanted kidneys and results in kidney injury and scarring. Presented here is a model of obstructive ...

a-murine-model-irreversible-reversible-unilateral-ureteric

Research

JoVE Journal

Medicine

30.3K vistas.  University of Edinburgh. The murine model of irreversible unilateral ureteric obstruction (UUO) is presented together with the model of reversible UUO in which the ureteric obstruction is relieved by anastomosis of the severed ureter into the bladder. These models enable the study of renal inflammation and scarring as well as tissue remodeling.

Video: A Murine Model of Irreversible and Reversible Unilateral Ureteric Obstruction

Creation of Reversible Cholestatic Rat Model

Cholestasis is a clinical condition commonly encountered by both surgeons and gastroenterologists. Cholestasis can cause various physiological changes and affect the nutritional status and surgical outcomes. Study of the pathophysiological changes occurring in the liver and other organs is of importance. Various studies have been done in cholestatic rat models.
We used a reversible cholestatic rat model in our recent study looking at the role of methylprednisolone in the ischemia reperfusion injury. Various techniques for creation of a reversible cholestatic model have been described. Creation of a reversible cholestatic rat model can be challenging in view of the smaller size and unique hepatopancreatobiliary anatomy in rats. This video article demonstrates the creation of a reversible cholestatic model.
This model can be used in various studies, such as looking at the changes in nutritional, physiological, pathological, histological and immunological changes in the gastrointestinal tract. This model can also be used to see the effects of cholestasis and various therapeutic interventions on major hepatic surgeries.

Cholestasis is a clinical condition commonly encountered by both surgeons and gastroenterologists. Creation of a reversible cholestatic rat model can be challenging in view of the smaller size and unique hepatopancreatobiliary anatomy in rats. This video article demonstrates the creation of a reversible cholestatic model.

Creación de un modelo reversible Rata colestásica

Cholestasis is a clinical condition commonly encountered by both surgeons and gastroenterologists. Cholestasis can cause various physiological changes and ...

Investigación

Medicina

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

Un modelo de la contundente unilateral Lesión Medular Cervical Uso del Impactador Infinite Horizon

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

Quantitative Real-Time PCR Evaluation of microRNA Expressions in Mouse Kidney with Unilateral Ureteral Obstruction

MicroRNAs (miRNAs) are single stranded, non-coding RNA molecules that typically regulate gene expression at the post-transcriptional level by binding to partially complementary target sites in the 3&#39; untranslated region (UTR) of messenger RNA (mRNA), which reduces the mRNA&#39;s translation and stability. The miRNA expression profiles in various organs and tissues of mice have been investigated, but standard methods for the purification and quantification of miRNA in mouse kidney have not been available. We have established an effective and reliable method for extracting and evaluating miRNA expression in mouse kidney with renal interstitial fibrosis by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The protocol required five steps: (1) creation of sham and unilateral ureteral obstruction (UUO) mice; (2) extraction of kidney samples from the UUO mice; (3) extraction of total RNA, which includes miRNA, from the kidney samples; (4) complementary DNA (cDNA) synthesis with reverse transcription from miRNA; and (5) qRT-PCR using the cDNA. Using this protocol, we successfully confirmed that compared to the controls, the expression of miRNA-3070-3p was significantly increased and those of miRNA-7218-5p and miRNA-7219-5p were significantly decreased in the kidneys of a mouse model of renal interstitial fibrosis. This protocol can be used to determine the miRNA expression in the kidneys of mice with UUO.

We describe a method for evaluating the microRNA expression in the kidneys of mice with unilateral ureteral obstruction (UUO) by quantitative reverse-transcription polymerase chain reaction. This protocol is suitable for studying kidney microRNA expression profiles in mice with UUO and in the context of other pathological conditions.

Evaluación cuantitativa de PCR en tiempo real de expresiones de microARN en riñón de ratón con obstrucción ureteral unilateral

MicroRNAs (miRNAs) are single stranded, non-coding RNA molecules that typically regulate gene expression at the post-transcriptional level by binding to ...

Genética

Nerve-sparing Mid-urethral Obstruction (NeMO) in Female Small Rodents

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal model to investigate bladder remodeling in pBOO are female rodents undergoing partial obstruction at the proximal urethra. Variability in the degree of obstruction and animal mortality are major concerns with proximal obstruction. Furthermore, dissecting around the proximal urethra and bladder neck jeopardizes bladder innervation.
We developed a nerve-sparing mid-urethral obstruction (NeMO) model for pBOO avoiding the disadvantages of the traditional model. We approached the urethra just inferior to the pubic symphysis, which obviated the need for laparotomy as well as for dissection in this area; also, the striated urethral sphincter remained untouched. We performed NeMO in female Sprague-Dawley rats (12 obstructions, 6 sham animals) as well as in female C57/bl6 mice (20 obstructions, 18 sham animals). After two weeks, we evaluated bladder function, bladder mass, and body mass.
We had no mortalities among obstructed- or sham-operated female rats; as described for the traditional proximal pBOO-method, we tied the suture around the proximal urethra and a temporarily placed 0.9 mm metal rod. NeMO induced an 85% increase in bladder mass after two weeks, average residual urine volume was 0.4 mL in partially obstructed rats while only 0.03 mL in sham animals. In mice, we tested 3 sizes of cannulas that we placed along the urethra when tying the suture. We found that using a 27-gauge cannula resulted in over 50% animal mortality; placing the 25-gauge cannula did not yield the desired response in increasing bladder mass; utilizing a 26-gauge cannula yielded favorable results with minimal animal mortality (1/8) yet a significant 2-fold increase in bladder mass.

Nerve-sparing Mid-urethral Obstruction NeMO in Female Small Rodents

Traditional modeling of partial bladder outlet obstruction in rodents is fraught with animal mortality. A denervation injury from dissection around the proximal urethra and bladder neck is also of major concern. We developed and evaluated a safe and reliable mid-urethral obstruction model, avoiding the shortcomings of the traditional model.

Preservación de los nervios de mitad de obstrucción uretral (NEMO) en femenino pequeños roedores

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal ...

Detrusor Underactivity Model in Rats by Conus Medullaris Transection

The goal of the presented protocol was to establish a detrusor underactivity (DU) model in the rat through conus medullaris transection. Laminectomy was performed in a total of 40 female Wistar rats (control group: 10 rats; test group: 30 rats) weighing 200&#8211;220 g, and the conus medullaris was transected at the L4&#8210;L5 level in the test group. All the rats were housed and fed under the same environmental conditions for six weeks. In the test group, urine voiding was performed twice daily for six weeks, and mean residual urine volume was recorded. A cystometrogram was performed in both groups. Maximum cystometric capacity (MCC), detrusor opening pressure (DOP), and compliance of the bladder were recorded and calculated. The test group showed significant urinary retention after the surgery, both during and after the spinal shock. However, no abnormality was observed in the control group. When compared to the control group, the MCC and compliance of bladder in the test group was significantly higher than that of the test group (3.24 &#177; 2.261 mL versus 1.04 &#177; 0.571 mL; 0.43 &#177; 0.578 mL/cmH2O versus 0.032 &#177; 0.016 mL/cmH2O), whereas DOP in the test group was lower than control (20.28 &#177; 14.022 cmH2O versus 35 &#177; 13.258 cmH2O). This method of establishing an animal model of DU by the conus medullaris transection offers an excellent opportunity to understand DU&#8217;s pathophysiology in a better manner.

We present a method for establishing a detrusor underactivity model by conus medullaris transection in rats. Detrusor underactivity was successfully stimulated in these animals. The model can be used for studying urinary tract function.

Modelo de hipoactividad del detrusor en ratas por transección del cono medullaris

The goal of the presented protocol was to establish a detrusor underactivity (DU) model in the rat through conus medullaris transection. Laminectomy was ...

Neurociencias

An Effective Mouse Model of Unilateral Renal Ischemia-Reperfusion Injury

Ischemia-reperfusion injury (IRI) is the leading cause of acute renal failure and is a significant contributor to delayed graft function. Animal models are the only available resources that mimic the complexities of the IRI-associated damage encountered in vivo. This paper describes an effective mouse model of unilateral renal IRI that delivers highly reproducible data. Ischemia is induced by occluding the right renal pedicle for 30 min followed by reperfusion. In addition to the surgical procedure, a sequential overview of the expected physiological and histopathological changes following renal IRI will be provided by comparing data from seven different reperfusion times (4 h, 8 h, 16 h, 1 day, 2 days, 4 days, and 7 days). Critical data for planning experiments ahead, such as mean surgical time, average anesthetic consumption, and body weight changes over time, will be shared. This work will help researchers implement a reliable renal IRI model and select the appropriate reperfusion time that aligns with their intended investigative goals.

Renal ischemia-reperfusion injury is associated with high morbidity and mortality in hospitalized patients. Here, we present a simple and effective mouse model of unilateral renal ischemia-reperfusion injury and provide a sequential overview of representative pathological changes observed in the kidney.

Un modelo de ratón eficaz de lesión unilateral por isquemia-reperfusión renal

Ischemia-reperfusion injury (IRI) is the leading cause of acute renal failure and is a significant contributor to delayed graft function. Animal models ...

Using 2-Photon Microscopy to Quantify the Effects of Chronic Unilateral Ureteral Obstruction on Glomerular Processes

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface glomeruli provide a unique opportunity to investigate physiological and pathophysiological processes using intravital 2-photon microscopy. Quantification of glomerular capillary blood flow and vasoconstriction and dilatation in response to drugs, permeability, and inflammation are just some of the processes that can be studied. In addition, transgenic rats, i.e., podocytes labeled with fluorescent dyes and other molecular biomarker approaches, provide increased resolution to directly monitor and quantify protein-protein interactions and the effects of specific molecular alterations.
In mice, which lack surface glomeruli after four weeks of age, unilateral ureteral obstruction (UUO) for several weeks has been used to induce surface glomeruli. As this induction model does not allow for baseline studies, we quantified the effects of UUO on glomerular processes in the UUO model in Munich Wistar Fr&#246;mter (MWF) rats, which have surface glomeruli under physiologic conditions. The UUO model for five weeks or more induced significant alterations to gross renal morphology, the peritubular and glomerular microvasculature, as well as the structure and function of tubular epithelia. Glomerular and peritubular red blood cell (RBC) flow decreased significantly (p &#60; 0.01), probably due to the significant increase in the adherence of white blood cells (WBCs) within glomerular and peritubular capillaries. The glomerular sieving coefficient of albumin increased from 0.015 &#177; 0.002 in untreated MWFs to 0.045 &#177; 0.05 in 5-week-old UUO MWF rats. Twelve weeks of UUO resulted in further increases in surface glomerular density and glomerular sieving coefficient (GSC) for albumin. Fluorescent albumin filtered across the glomeruli was not reabsorbed by the proximal tubules. These data suggest that using UUO to induce surface glomeruli limits the ability to study and interpret normal glomerular processes and disease alterations.

Here, we present a protocol using 2-photon microscopy in Munich Wistar Fromter rats with surface glomeruli to quantifythe effects of prolonged ureteral obstruction on glomerular dynamics and function.

Uso de microscopía de 2 fotones para cuantificar los efectos de la obstrucción ureteral unilateral crónica en los procesos glomerulares

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface ...

An Immature Murine Model of Reversible Unilateral Ureteral Obstruction

Unilateral ureteral obstruction (UUO) is a common cause of chronic kidney disease (CKD), leading to the progression of renal interstitial fibrosis and ultimately resulting in irreversible kidney damage. The alleviation of UUO is crucial. Several animal models of reversible unilateral ureteral obstruction (RUUO) have been established in the literature, enabling the observation of structural changes and functional damage while also simulating physiological and pathophysiological changes following the relief of ureteral obstruction. In this study, a reversible obstruction model was established in the unilateral murine ureter using a silicone tube. Significant renal damage was observed prior to obstruction relief, with partial recovery noted afterward. Unlike UUO, this model prevents progressive hydronephrosis, leading to distinct pathological outcomes. This simple surgical procedure demonstrates a high success rate and holds promise as a classical model for investigating reversible obstructive nephropathy and potential treatments for renal interstitial fibrosis. Furthermore, it provides a practical platform for studying the mechanisms of recovery from obstructive nephropathy, renal cell regeneration, and tissue remodeling.

The present protocol describes a step-by-step, reproducible model of unilateral ureteral obstruction.

Un modelo murino inmaduro de obstrucción ureteral unilateral reversible

Unilateral ureteral obstruction (UUO) is a common cause of chronic kidney disease (CKD), leading to the progression of renal interstitial fibrosis and ...

Unilateral Ureteral Obstruction Model for Investigating Kidney Interstitial Fibrosis

Kidney fibrosis is the final pathological outcome of progressive chronic kidney disease (CKD). The unilateral ureteral obstruction (UUO) model is widely used to elucidate the molecular and cellular mechanisms underlying kidney interstitial fibrosis and to identify potential therapeutic targets. This model is established in mice through surgical ligation of a unilateral ureter, a procedure that is relatively straightforward and easy to perform. However, the UUO mouse model is known to exhibit significant variability and inconsistency, influenced by factors such as mouse strain, age, sex, anesthesia type, duration of surgery, body temperature during the procedure, the operator's surgical skills, feeding conditions, and the overall health status of the mice. Variations in surgical techniques, suture placement, and the duration of obstruction contribute to the variability in outcomes. Additionally, inconsistent sampling of obstructed kidneys further increases variability in the assessment of kidney fibrosis. This study outlines the process of developing the UUO mouse model and evaluating interstitial fibrosis, discusses the technical challenges contributing to the model's unpredictability, and proposes potential solutions. These insights aim to establish a more standardized and universally applicable approach for investigating kidney fibrosis.

This protocol describes a method for inducing unilateral ureteral obstruction (UUO) in mice to study the progression of tissue fibrosis in obstructive nephropathy. It includes surgical procedures, post-operative care, and methods for fibrosis assessment.

Kidney fibrosis is the final pathological outcome of progressive chronic kidney disease (CKD). The unilateral ureteral obstruction (UUO) model is widely ...

Unilateral Ureteral Obstruction Murine Model: A Surgical Procedure to Obstruct the Urinary Flow from a Kidney in Mouse Models

Source: Yanai, K. et al. <a href="https://www.jove.com/video/61383" target="_blank">Quantitative real-time PCR evaluation of microRNA expressions in mouse kidney with unilateral ureteral obstruction.</a> J. Vis. Exp. (2020)
This video demonstrates the procedure to generate a unilateral ureteral obstruction mouse model. The procedure involves cutting one of the ureters of the mouse to obstruct urine flow.

Modelo Murino de Obstrucción Ureteral Unilateral: Un Procedimiento Quirúrgico para Obstruir el Flujo Urinario de un Riñón en Modelos de Ratón

Source: Yanai, K. et al. Quantitative real-time PCR evaluation of microRNA expressions in mouse kidney with unilateral ureteral obstruction. J. Vis. Exp. ...